Using specific cell lines we confirmed that the glucocorticoid receptor from New World primates has a lower binding affinity than the human receptor (apparent Kd, 20.9 versus 4.3 nM, respectively). As a first step in understanding the mechanism of this lower affinity in New World primates, we used reverse transcriptase-PCR to clone the glucocorticoid receptor from squirrel monkey and have compared the sequence to receptor sequences obtained from owl monkey, cotton-top tamarin and human cells. The squirrel monkey glucocorticoid receptor is approximately 97% identical in nucleotide and amino acid sequence to the human receptor (pub 2). The ligand binding domain of the squirrel monkey glucocorticoid receptor contains four amino acid differences, all of which are present in owl monkey and cotton-top tamarin receptors. The DNA-binding domain is completely conserved among all four receptors. Twenty-two differences from the human sequence were found in the N-terminal region of the squirrel monkey receptor. None of the substitutions in the ligand binding domain matched mutations known to influence binding affinity in other species. To determine whether these substitutions were responsible for decreased affinity, squirrel monkey and human receptors were expressed in the TNT Coupled Reticulocyte Lysate System. Expressions of human and squirrel monkey glucocorticoid receptors and a squirrel monkey receptor in which Phe774 was mutated to Leu (F774L) were similar. When expressed in the TNT system, the squirrel monkey and human glucocorticoids had similar, high affinity binding for dexamethasone (apparent Kd, 5.9 and 4.3 nM, respectively), whereas the squirrel monkey F774L receptor had lower affinity binding (apparent Kd, 20.4 nM). Thus, substitutions in the ligand-binding domain of the squirrel monkey glucocorticoid receptor cannot account for the decreased binding affinity of these receptors in squirrel monkey cells.